Mass spectrometric assays for peptides

ABSTRACT

Methods for interpretation of mass spectrometric tests for clinical biomarkers in which the amounts of internal standards are set to equal clinical evaluation thresholds, and preparations for adding stable isotope labeled peptide species to sample digests while minimizing losses and alterations in peptide stoichiometry.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. patent application61/314,149, entitled “MS Internal Standards at Clinical Levels” filed onMar. 15, 2010 and U.S. patent application 61/314,154, entitled “StableIsotope Labeled Peptides on Carriers” filed on Mar. 15, 2010; thedisclosures of each of which are herein incorporated by reference intheir entirety.

The disclosures of U.S. patent application Ser. No. 10/676,005, entitledHigh Sensitivity Quantitation of Peptides by Mass Spectrometry; filed 2Oct. 2003; Ser. No. 60/415,499, entitled Monitor Peptide EnrichmentUsing Anti-Peptide Antibodies, filed 3 Oct. 2002; Ser. No. 60/420,613,entitled Optimization of Monitor Peptide Enrichment Using Anti-PeptideAntibodies, filed 23 Oct. 2002; Ser. No. 60/449,190, entitled HighSensitivity Quantitation of Peptides by Mass Spectrometry, filed 20 Feb.2003; Ser. No. 60/496,037, entitled Improved Quantitation of Peptides byMass Spectrometry, filed 18 Aug. 2003; Ser. No. 60/557,261, entitledSelection of Antibodies and Peptides for Peptide Enrichment, filed 29Mar. 2004; Ser. No. 11/256,946, entitled Process For Treatment OfProtein Samples, filed 25 Oct. 2005, describing methods for processingprotein samples imbibed within a porous membrane; Ser. No. 12/042,931,entitled Magnetic Bead Trap and Mass Spectrometer Interface”; and Ser.No. 11/147,397, entitled Stable Isotope Labeled Polypeptide Standardsfor Protein Quantitation” describing means for production of labeledpeptide internal standards as recombinant concatamer proteins filed 8Jun. 2005, are each herein incorporated by reference in theirentireties.

U.S. Pat. No. 6,649,419, entitled Method and Apparatus for ProteinManipulation, filed 28 Nov. 2000, describing methods of eluting analytescaptured on magnetic beads is herein incorporated by reference in theirentireties.

BACKGROUND

Methods of using mass spectrometry for the measurement of establishedand candidate biomarker proteins could benefit from improvements inmethods for the preparation, handling and quantitative performance ofstable isotope labeled peptides. Such labeled peptides are used asinternal standards in the analysis of peptides samples, includingprotein sample digests, by mass spectrometry. In a specific case, theart could benefit from improvements to the performance of the technologycalled “SISCAPA” that was disclosed in one or more of the patent filingsreferenced above, and a number of recent publications including, forinstance: An effective and rapid method for functional characterizationof immunoadsorbents using POROS® beads and flow cytometry, N. LeighAnderson et al., Journal of Proteome Research 3:228-34 (2004); MassSpectrometric Quantitation of Peptides and Proteins Using Stable IsotopeStandards and Capture by Anti-Peptide Antibodies (SISCAPA), Anderson, N.L. et al., Journal of Proteome Research 3: 235-44 (2004); Anti-peptideantibody screening: selection of high affinity monoclonal reagents by arefined surface plasmon resonance technique, Matthew E. Pope et al., J.Immunol. Methods, 341(1-2):86-96 (2009); A Human Proteome Detection andQuantitation Project: hPDQ, N. Leigh Anderson, et al., Mol. Cell.Proteomics 8:883-886 (2009); SISCAPA Peptide Enrichment on MagneticBeads Using an Inline Beadtrap Device, N. Leigh Anderson, et al., Mol.Cell. Proteomics 8:995-1005 (2009); An automated and multiplexed methodfor high throughput peptide immunoaffinity enrichment and multiplereaction monitoring mass spectrometry-based quantification of proteinbiomarkers, Jeffrey R. Whiteaker, et al., Mol. Cell. Proteomics9:184-196 (2010); Proteomic-Based Multiplex Assay Mock Submissions:Supplementary Material to A Workshop Report by the NCI-FDA InteragencyOncology Task Force on Molecular Diagnostics, Fred E. Regnier, et al.,Clin. Chem. 56:2 165-171 (2010); MALDI Immunoscreening (MiSCREEN): AMethod for Selection of Anti-peptide Monoclonal Antibodies for Use inImmunoproteomics, Morteza Razavi, Matthew E. Pope, Martin V. Soste,Brett A. Eyford, N. Leigh Anderson and Terry W. Pearson, Journal ofImmunological Methods 364:50-64 (2011).

SUMMARY

Quantitative assays for evaluation of proteins in complex samples areprovided, including assays of clinical specimens such as human plasmaand other proteinaceous samples (including for example tissues,secretions, and body fluids of all living things, as well as samplesprepared from heterogeneous mixtures of these), and specifically to thegeneration and use of stable isotope labeled peptides as Stable IsotopeStandards (SIS) in assays based on mass spectrometric readouts.

Internal standards are provided, against which clinically importantproteins and peptides derived from them by digestion can be compared.The internal standards are added to test samples at known concentrationsthat can, for example, be equivalent to clinical decision levels for therespective analytes, thereby facilitating interpretation of test resultsthrough the direct comparison of analyte amount with the amount of aninternal standard as measured by a mass spectrometer (e.g., as peptidesignal peak height or peak area). One internal standard suffices in acase where a single threshold (“test cutoff”) value is used in resultinterpretation (e.g., in measurements of prostate specific antigen),while two internal standards may be used where a normal range is usedwith upper and lower bounds (one standard provided at the lower boundconcentration and the other at the higher bound concentration). Usinginternal standards in this manner enables simplified testinterpretation. The approach can be generalized to provide internalstandards set to personalized reference values, allowing precisecomparison of an individual to his/her own historical reference value orrange. The approach may also use 2, 3, 4, 5, 6 or more internalstandards at different concentrations, wherein at least one standard ispresent at the threshold value used for results interpretation where asingle threshold (“test cutoff”) value is used in result interpretation.Alternatively, the approach may also employ 3, 4, 5, 6 or more internalstandards at different concentrations, wherein standards are present atthe upper and lower threshold values used for results interpretationwhere a normal range is used with upper and lower bounds.

The technology also provides methods for the production, purification,characterization and use of stable-isotope-labeled peptide sequenceswhich can be used together or separately as internal standards in themass spectrometric quantitation of peptides and proteins. Briefly, oneor more monitor peptide sequences (the “analytes”) are selected torepresent each protein to be measured. In the case of trypsin cleavageof the analyte-containing sample, candidate monitor peptides will betryptic peptides (i.e., generally ending in K or R). A set of selectedmonitor peptide sequences representing multiple protein analytes canthen be synthesized, each with a sequence extension that: 1) provides achemical linkage site capable of joining the peptide to a large molecule“carrier”; and 2) establishes a proteolytic enzyme cleavage site withinthe peptide such that cleavage at the site releases from the carrier alabeled peptide of the same sequence as the intended analyte peptide.Stable isotopes (particularly ¹⁵N or ¹³C isotopically-enriched to >98%isotopic purity, but also ²H, ¹⁸O and any other stable isotopes ofelements independently present in proteins) can be incorporated duringsynthesis by chemical means, in which case the stable isotopes are eachindependently incorporated at >95%, 96%, >97% or >98% substitution for anatural isotope at specific positions in the structure of amino acids ortheir analogous peptide synthesis precursors. Stable isotopes can alsobe incorporated into peptides by in vivo or in vitro means, in whichcase an organism or an in vitro translation system can be supplied withmetabolic precursors or amino acids labeled at >95%, 96%, >97% or >98%isotopic substitution to achieve a highly substituted peptide product.

One or more labeled peptides can be covalently linked via chemicallinkage to a carrier molecule such as keyhole limpet hemocyanine (KLH)to provide a stable, easily quantitated source of labeled peptide. Theresulting “carrierSIS” molecule is substantially more easily handledthan the labeled peptide alone, in part because it is highly solubleeven after binding of many copies of the SIS peptide and thus is lesslikely to stick to surfaces of storage vials, pipettes, etc., than is afree SIS peptide. The carrierSIS protein can be purified using specifictags incorporated into the carrier molecule (e.g., peptide or biotintags) or based on physical properties such as solubility or size (i.e.,on an SDS electrophoresis gel). The intact carrierSIS protein can bequantitated once by amino acid analysis, which together with a knowledgeof the quantity of bound SIS peptide yields a molar concentration thatapplies to SIS peptides subsequently liberated by proteolysis.

The carrierSIS protein can be added at known amounts to complex proteinsamples prior to proteolytic digestion, and digested with the sampleproteins to produce a series of SIS peptides whose stoichiometry to oneanother is known, and whose absolute concentration is also known.Alternatively the carrier in the carrierSIS can be a particle containingnon-protein components (e.g., a magnetic bead). In use the carrierSIScan be digested with the sample to liberate SIS peptide or pre-digestedto yield a stoichiometric mixture of SIS peptides to be added to asample before or after sample digestion. These SIS peptides are thenused as standards for quantitation of sample protein derived peptides bymass spectrometry (e.g., as in the SISCAPA method disused in U.S. Pat.No. 7,632,686, entitled “High Sensitivity Quantitation of Peptides byMass Spectrometry”). The carrierSIS construct provides an alternative tothe previously disclosed recombinant method for producing SIS-containingconcatamer proteins (U.S. patent application Ser. No. 11/147,397 “StableIsotope Labeled Polypeptide Standards for Protein Quantitation”).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Comparison of analyte and internal standard peptides byMALDI-MS. Internal standard 1 is present in an amount such that itssignal level 3 indicates the clinical decision point for the endogenousanalyte 2.

FIG. 2. Comparison of analyte and two internal standard peptides byMALDI-MS. One internal standard 1 is present at a concentration equal tothe lower limit of the analyte reference interval 3, and the other 4 ispresent at the upper limit of the interval 5.

FIG. 3. Comparisons of analyte and internal standard amounts by selectedreaction monitoring LC-MS/MS.

FIG. 4. Comparison of analyte and two internal standard peptides byselected reaction monitoring LC-MS/MS.

FIG. 5. MALDI comparisons of analyte and SIS peptide levels.

FIG. 6. Linkage to and release of extended SIS peptides from anactivated carrier molecule. Extension sequence CGSGK SEQ ID No. 6,extended peptides CGSGKNFPSPVDAAFR and SEQ ID No. 8, CGSGKEIGELYPK SEQID No. 6, and released peptides NFPSPVDAAFR SEQ ID No. 7, and EIGELYLPKSEQ ID No. 5.

FIG. 7. Linkage of a polySIS peptide to an activated carrier and releaseof SIS peptides by tryptic digestion. The sequence of the polySIS isdescribed in detail as Seq ID No. 39 in U.S. patent application Ser. No.11/147,397. Part A shows the extension sequences GSGC SEQ ID No. 9 thatcan be linked to a carrier (41) by an optional spacer (42) that has acarrier linkage site denoted “M” (43), along with the polySIS sequence(46) that may bear carboxyl terminal extension sequence (45) having aterminal cysteine (44):

SEQ ID No. 11MSGSHHHHHHSSGIEGRGRLIKHMTMAKATEHLSTLSEKNWGLSVYADKPETTKILGGHLDAKDTVQIHDITGKTVIGPDGHKQGFGNVATNTDGKEIGELYLPKTGLQEVEVKDDLYVSDAFHKIYHSHIDAPKETAASLLQAGYKITQVLHFTKFPEVDVLTKLGNQEPGGQTALKLSSPAVITDKQWAGLVEKIPPWEAPKLFLEPTQADIALLKSHAPEVITSSPLKIFYNQQNHYDGSTGKEHSSLAFWKVSVSQTSKESDTSYVSLKWELDLDIKSTVLTIPEIIIKLIENGYFHPVKASYPDITGEKDPPSDLLLLKALQDQLVLVAAKAEIEYLEKQPGGIRGSGC,;

Part B, shows the polySIS sequences including extension sequences (47)that are bound to the carrier.

Part C shows a collection of the peptides (48) released followingproteolytic treatment of the carrier leaving the extension sequence(where one is employed) attached to the carrier in this example.

MSGSHHHHHHSSGIEGR,; SEQ ID No. 12 GRLIK,; SEQ ID No. 13 HMTMAK,;SEQ ID No. 14 ATEHLSTLSEK,: SEQ ID No. 15 NWGLSVYADKPETTK,;SEQ ID No. 16 ILGGHLDAK,; SEQ ID No. 17 DTVQIHDITGK,; SEQ ID No. 18TVIGPDGHK,; SEQ ID No. 19 QGFGNVATNTDGK,; SEQ ID No. 20 EIGELYLPK,;SEQ ID No. 21 TGLQEVEVK,; SEQ ID No. 22 DDLYVSDAFHK,; SEQ ID No. 23IYHSHIDAPK,; SEQ ID No. 24 ETAASLLQAGYK,; SEQ ID No. 25 ITQVLHFTK,;SEQ ID No. 26 FPEVDVLTK,; SEQ ID No. 27 LGNQEPGGQTALK,; SEQ ID No. 28LSSPAVITDK,; SEQ ID No. 29 QWAGLVEK,; SEQ ID No. 30 IPPWEAPK,;SEQ ID No. 31 LFLEPTQADIALLK,; SEQ ID No. 32 SHAPEVITSSPLK,;SEQ ID No. 33 IFYNQQNHYDGSTGK,; SEQ ID No. 34 EHSSLAFWK,; SEQ ID No. 35VSVSQTSK,; SEQ ID No. 36 ESDTSYVSLK,; SEQ ID No. 37 WELDLDIK,;SEQ ID No. 38 STVLTIPEIIIK,; SEQ ID No. 39 LIENGYFHPVK,; SEQ ID No. 40ASYPDITGEK,; SEQ ID No. 41 DPPSDLLLLK,; SEQ ID No. 42 ALQDQLVLVAAK,;SEQ ID No. 43 AEIEYLEK,; SEQ ID No. 44 and QPGGIR,; SEQ ID No. 45

DETAILED DESCRIPTION

The term “amount”, “concentration” or “level” of an analyte or internalstandard means the physical quantity of the substance referred to,either in terms of mass (or equivalently moles) or in terms ofconcentration (the amount of mass or moles per volume of a solution orliquid sample).

The term “analyte” or “ligand” may be any of a variety of differentmolecules, or components, pieces, fragments or sections of differentmolecules that are to be measured or quantitated in a sample. An analytemay thus be a protein, a peptide derived from a protein by digestion orother fragmentation technique, a small molecule (such as a hormone, ametabolite, a drug, a drug metabolite) or nucleic acids (DNA, RNA, andfragments thereof produced by enzymatic, chemical or other fragmentationprocesses).

The term “antibody” means a monoclonal or monospecific polyclonalimmunoglobulin protein such as IgG or IgM. An antibody may be a wholeantibody or antigen-binding antibody fragment derived from a species(e.g., rabbit or mouse) commonly employed to produce antibodies againsta selected antigen, or may be derived from recombinant methods such asprotein expression, and phage/virus display. See, e.g., U.S. Pat. Nos.7,732,168; 7,575,896; and 7,431927, which describe preparation of rabbitmonoclonal antibodies. Antibody fragments may be any antigen-bindingfragment that can be prepared by conventional protein chemistry methodsor may be engineered fragments such as scFv, diabodies, minibodies andthe like. It will be understood that other classes of molecules such asDNA and RNA aptamers configured as specific and high affinity bindingagents may, be used as alternatives to antibodies or antibody fragmentsin appropriate circumstances.

The term “bind” or “react” means any physical attachment or closeassociation, which may be permanent or temporary. Generally, reversiblebinding includes aspects of charge interactions, hydrogen bonding,hydrophobic forces, van der Waals forces etc., that facilitate physicalattachment between the molecule of interest and the analyte beingmeasuring. The “binding” interaction may be brief as in the situationwhere binding causes a chemical reaction to occur. Reactions resultingfrom contact between the binding agent and the analyte are also withinthe definition of binding for the purposes of the present technology,provided they can be later reversed to release a monitor fragment.

The term “binding agent” means a molecule or substance having anaffinity for one or more analytes, and includes antibodies (for examplepolyclonal, monoclonal, single chain, and modifications thereof),aptamers (made of DNA, RNA, modified nucleotides, peptides, and othercompounds), etc. “Specific binding agents” are those with particularaffinity for a specific analyte molecule.

The terms “clinical evaluation threshold”, “clinical decision point” and“test evaluation threshold” (which are used here interchangeably) mean avalue of analyte abundance or concentration in a biological (includingclinical) sample that serves as a decision point for a test result(typically a clinical test result). For example, a threshold of 4.1ng/ml has been used as a clinical decision point in the interpretationof prostate specific antigen test results in men: values above thislevel indicate increased risk of prostate cancer, and below this levelno action in typically taken. Alternatively for some analytes a valuelower than the clinical decision point can be the indication of higherrisk. Clinical decision points are typically derived from large clinicalstudies and represent a statistical parameter set by clinicalresearchers to provide a given specificity and sensitivity in answeringa particular clinical question. Similarly, methods of risk analysis canbe used to determine test evaluation thresholds in non-clinicalsituations, such as the determination of permitted levels of certainprotein or peptide contaminants in foods, beverages, drugs, cosmetics,environmental and other samples required to address regulatoryrequirements in various countries. For use in a specific test it will beunderstood that a test evaluation threshold can be set based on resultsobtained using the specific test or an equivalent methodology, in orderthat any analytical biases inherent in the test be reflected in thethreshold. Thus, if an MS-based assay detects a peptide obtained bytryptic digestion of a target protein in a sample, and for some reasonthe tryptic yield of this peptide is reproducible but not 100%, forexample an 85% yield of peptide per protein on a molar basis, then atest evaluation threshold for this peptide in the example would be set avalue equivalent to 85% of the amount of the corresponding protein inthe sample, i.e., equal to the level of peptide analyte observed in theMS at the test evaluation threshold.

The terms “clinical reference range” and “clinical reference interval”mean the range of abundance or concentration values of an analyte thatare deemed to be with the “normal” clinical range. Such ranges arefrequently established by determination of analyte levels in a normalpopulation, and the clinical reference range typically determined as thecentral 95% of the resulting histogram (with 2.5% of the populationabove and 2.5% below the resulting high and low values). As used here,these terms also refer to ranges whose bounds are defined by clinicalfeatures other than the distribution of results in normal individuals(e.g., the population reference range in diabetic patients), andclinical ranges based on a patient's prior test values for the same orother analytes, alone or in combination with population test data. Avariety of statistical approaches can be used to calculate such rangesfrom analyte measurements, and this is advantageously can be done priorto their application in the design of an assay or the determination ofan amount of internal standard to use in the assay. As in the case of asingle test evaluation threshold, it will be understood that a clinicalreference interval for use in a specific test can be set based onresults obtained using the specific test or an equivalent methodology,in order that any analytical biases inherent in the test are reflectedin the threshold.

The terms “clinical cutpoint panel” and “multi-threshold classifier”mean a set of two or more different threshold levels used to assess theresult of an assay by comparing a measure of analyte amount with suchthreshold levels to determine whether analyte amount is higher than thehighest threshold, lower than the lowest threshold, or between twoparticular threshold levels, and include clinical interpretive scaleswith more than two references values (e.g., a three point scale whose 4segments indicate no, low, moderate or high risk).

The term “carrier linkage site” refers to a chemical grouping or moietyin a suitably prepared carrier molecule that is capable of reacting witha peptide linkage site in a modified or unmodified peptide molecule toyield a covalent or very tight non-covalent linkage between the peptideand the carrier. In one embodiment, the chemical linkage site is presentwithin the extension to the SIS peptide.

The term “carrier molecule” refers to a molecule having multiple sitesat which peptides can be attached, said attachment being either covalentor non-covalent but tight (e.g., via avidin-biotin interaction).Examples of carriers include proteins (e.g., activated KLH or albuminbearing multiple maleimide groups [e.g., Thermofisher ImjectMaleimide-Activated Mariculture KLH] capable of reacting with multiplecysteine-containing SIS peptides), and other polymeric molecules havingdesirable properties such as solubility and stability, including forexample dextrans, linear polyacrylamides, agarose, dendrimers, etc.Carrier molecules are typically larger than tryptic peptides, generallyhaving molecular weights in the range 3,000 to 20,000,000. In someembodiments (e.g., polymer carriers) the carrier molecules will havemolecular weights in a range: from about 3,000 to about 10,000, or fromabout 10,000 to about 100,000, or from about 100,000 to about 1,000,000,or from about 1,000,000 to about 5,000,000, or from about 5,000,000 toabout 20,000,000 Daltons.

The term “carrier particle” means a physical particle having sites(e.g., single or multiple sites) at which peptides can be attached, saidattachment being either covalent or non-covalent and of a kind such thatthe peptide may released from the particle at an appropriate point inthe sample preparation workflow. Examples include agarose beads,magnetic beads, quantum dots, viruses, and particles of controlled-poreglass, chromatography media such as POROS or silica, and polystyrene.Carrier particles can be in the range of 10 nanometer to 1 millimeter insize. In some embodiments the carrier particles will have a size (e.g.,average diameter) in a range: from about 10 to about 100, or from about100 to about 10,000, or from about 10,000 to about 100,000, or fromabout 10,000 to about 1,000,000, or from about 100 to about 10,000, orfrom about 1,000 to about 100,000 nanometers.

The term “carrier surface” means a surface to which peptides can beattached, said attachment being either covalent or non-covalent and of akind such that the peptide is released from the particle at theappropriate point in the sample preparation workflow. Examples includethe surfaces (often the inner liquid-contacting surfaces) of vesselssuch as sample wells, pipette tips, cuvettes, or capillary tubes used ina sample processing workflow.

The term “carrier” means a carrier molecule, a carrier particle or acarrier surface.

The term “carrierSIS” or “conjugate” or “carrierSIS complex” or “SISpeptide delivery vehicle” means a carrier to which SIS peptides orextended SIS peptides are bound or linked.

The term “carrierPolySIS” or “conjugate” or “carrierPolySIS complex” or“polySIS peptide delivery vehicle” means a carrier to which polySISpeptides or extended polySIS peptides are bound or linked.

The term “denaturant” includes a range of chaotropic and other chemicalagents that act to disrupt or loosen the 3-D structure of proteinswithout breaking covalent bonds, thereby rendering them more susceptibleto proteolytic treatment. Examples include urea, guanidinehydrochloride, ammonium thiocyanate, trifluoroethanol and deoxycholate,as well as solvents such as acetonitrile, methanol and the like.

The term “electrospray ionization” (ESI) refers to a method for thetransfer of analyte molecules in solution into the gas and ultimatelyvacuum phase through use of a combination of liquid delivery to apointed exit and high local electric field.

The term “extended peptide” means a peptide having a subsequence that isthe same as a peptide analyte and one or more additional subsequencesand chemical moieties that provide additional functionality (such asattachment to a carrier) but are separated from the analyte subsequenceby action of the proteolytic activity used to create a sample digest.

The term “extended SIS” means an extended peptide comprising a labeledpeptide segment that is a SIS peptide.

The term “immobilized enzyme” means any form of enzyme that is fixed tothe matrix of a support by covalent or non-covalent interaction suchthat the majority of the enzyme remains attached to the support of themembrane.

The term “magnet”, “permanent magnet”, or “electromagnet” are used hereto mean any physical system, whether electrically powered or static,capable of generating a magnetic field.

The term “magnetic field” or “magnetic field gradient” are used hereinterchangeably, and refer to a physical region within which a spatiallyvarying magnetic field exists.

The terms “magnetic particle” and “magnetic bead” are usedinterchangeably and mean particulate substances capable of carryingbinding agents (whether attached covalently or non-covalently,permanently or temporarily) and which can respond to the presence of amagnetic field gradient by movement. The term includes beads that arereferred to as paramagnetic, superparamagnetic, and diamagnetic.

The terms “particle” or “bead” mean any kind of particle in the sizerange between 10 nm and 1 cm, and includes magnetic particles and beads.

The term “MALDI” means Matrix Assisted Laser Desorption Ionization andrelated techniques such as SELDI, and includes any technique thatgenerates charged analyte ions from a solid analyte-containing materialon a solid support under the influence of a laser or other means ofimparting a short energy pulse.

The term “Mass spectrometer” (or “MS”) means an instrument capable ofseparating molecules on the basis of their mass m, or m/z where z ismolecular charge, and then detecting them. In one embodiment, massspectrometers detect molecules quantitatively. An MS may use one, two,or more stages of mass selection. In the case of multistage selection,some means of fragmenting the molecules is typically used betweenstages, so that later stages resolve fragments of molecules selected inearlier stages. Use of multiple stages typically affords improvedoverall specificity compared to a single stage device. Often,quantitation of molecules is performed in a triple-quadrupole massspectrometer, but it will be understood herein that a variety ofdifferent MS configurations may be used to analyze the moleculesdescribed, and specifically MALDI instruments including MALDI-TOF,MALDI-TOF/TOF, and MALDI-TQMS and electrospray instruments includingESI-TQMS and ESI-QTOF, in which TOF means time of flight, TQMS meanstriple quadrupole MS, and QTOF means quadrupole TOF.

The term “monitor fragment” may mean any piece of an analyte up to andincluding the whole analyte that can be produced by a reproduciblefragmentation process (or without a fragmentation if the monitorfragment is the whole analyte) and whose abundance or concentration canbe used as a surrogate for the abundance or concentration of theanalyte. The term “monitor peptide” or “target peptide” means a peptidechosen as a monitor fragment of a protein or peptide.

The terms “multiplex clinical thresholds” or “multiplex test thresholds”mean a series of test values of analyte abundance or concentration in abiological (including clinical) sample that serve as decision points fora series of individual analyte measurements carried out as a single testprocedure (i.e., in a multiplex format). In the simplest case, aspecific test evaluation threshold is provided for each analyte in thepanel, and the result of the multiplex test is determined by analgorithm taking into account which analytes exceed and which do notexceed their respective thresholds.

The term “Natural” or “Nat” means the form of such a peptide that isderived from a natural biological sample by proteolytic digestion, andthus, contains approximately natural abundances of elemental isotopes.Nat peptides typically do not contain appreciable amounts of a stableisotope label such as is intentionally incorporated in SIS internalstandards.

The term “personal reference level” and “personal reference range” referto the use of analyte levels established previously for an individualpatient in the interpretation of test results.

The term “peptide-carrier conjugate” means a carrier with peptide(s)linked to it through bond(s) between carrier linkage site(s) and peptidelinkage site(s).

The term “peptide linkage site” refers to a chemical grouping or moietyin a modified or unmodified peptide molecule that is capable of reactingwith a carrier linkage site in a suitably prepared carrier molecule toyield a covalent or very tight non-covalent linkage between the peptideand the carrier. In one embodiment, the chemical linkage site is presentwithin the extension to the SIS peptide.

The term polySIS means a protein or peptide comprising subsequencesidentical to monitor peptides representing two or more differentproteins in sequence contexts such that the monitor peptides arereleased by proteolytic digestion.

The term “proteolytic enzyme cleavage site” refers to a site within anextended SIS peptide sequence at which the chosen proteolytic treatment(typically an enzyme such as trypsin) cleaves the extended SIS sequence,releasing peptides fragments (typically two) of which one is the SISpeptide sequence (identical to the analyte, or Nat, sequence for whichthe SIS serves as an internal standard).

The term “proteolytic treatment” or “enzyme” may refer any of a largenumber of different enzymes, including trypsin, chymotrypsin, lys-C, v8and the like, as well as chemicals, such as cyanogen bromide. In thiscontext, a proteolytic treatment acts to cleave peptide bonds in aprotein or peptide in a sequence-specific manner, generating acollection of shorter peptides (a digest).

The term “sample” means any complex biologically-generated samplederived from humans, other animals, plants or microorganisms, or anycombinations of these sources. “Complex digest” means a proteolyticdigest of any of these samples resulting from use of a proteolytictreatment.

The terms “SIS”, “stable isotope standard” and “stable isotope labeledversion of a peptide or protein analyte” mean a peptide or protein, suchas a peptide or protein having a unique sequence that is identical orsubstantially identical to that of a selected peptide or proteinanalyte, and including a label of some kind (e.g., a stable isotope)that allows its use as an internal standard for mass spectrometricquantitation of the natural (unlabeled, typically biologicallygenerated) version of the analyte (see U.S. Pat. No. 7,632,686 “HighSensitivity Quantitation of Peptides by Mass Spectrometry”). In oneembodiment, a SIS peptide or protein comprises a peptide sequence thathas a structure that is chemically identical to that of the molecule forwhich it will serve as a standard, except that it has isotopic labels atone or more positions that alter its mass. Hence a SIS is 1) recognizedas equivalent to the analyte in a pre-analytical workflow, and is notappreciably differentially enriched or depleted compared to the analyteprior to mass spectrometric analysis, and 2) differs from it in a mannerthat can be distinguished by a mass spectrometer, either through directmeasurement of molecular mass or through mass measurement of fragments(e.g., through MS/MS analysis), or by another equivalent means. Stableisotope standards include peptides having non-material modifications ofthis sequence, such as a single amino acid substitution (as may occur innatural genetic polymorphisms), substitutions (including covalentconjugations of cysteine or other specific residues), or chemicalmodifications (including glycosylation, phosphorylation, and otherwell-known post-translational modifications) that do not materiallyaffect enrichment or depletion compared to the analyte prior to massspectrometric analysis. In one embodiment, SIS are those in which thelevel of substitution of each stable isotope (e.g., ¹³C, ¹⁵N, ¹⁸O or ²H)at the specific sites within the peptide structure where the isotope(s)is/are incorporated (i.e., those sites that depart significantly fromthe natural unenriched isotope distribution) is/are >95%, >96%, >97%, or>98%.

The term “SISCAPA” means the method described in U.S. Pat. No.7,632,686, entitled High Sensitivity Quantitation of Peptides by MassSpectrometry and in Mass Spectrometric Quantitation of Peptides andProteins Using Stable Isotope Standards and Capture by Anti-PeptideAntibodies (SISCAPA). Anderson, N. L., Anderson, N. G., Haines, L. R.,Hardie, D. B., Olafson. R. W., and Pearson, T. W, Journal of ProteomeResearch 3: 235-44 (2004).

The term “small molecule” or “metabolite” means a multi-atom moleculeother than proteins, peptides and DNA; the term can include but is notlimited to amino acids, steroid and other small hormones, metabolicintermediate compounds, drugs, drug metabolites, toxicants and theirmetabolites, and fragments of larger biomolecules.

The term “stable isotope” means an isotope of an element naturallyoccurring or capable of substitution in proteins or peptides that isstable (does not decay by radioactive mechanisms) over a period of a dayor more. The primary examples of interest in this context are ¹³C, ¹⁵N,²H, and ¹⁸O, of which the most commonly used are ¹³C and ¹⁵N.

The term “standardized sample” means a protein or peptide sample towhich stable isotope labeled version(s) of one or more peptide orprotein analytes have been added at levels corresponding to testevaluation thresholds to serve as internal standards.

The following embodiments of the present technology make use of a seriesof concepts described in this specification. These concepts providebackground as to specific embodiments of the methods and compositionsdescribed herein.

1 The SISCAPA Method

SISCAPA assays combine affinity enrichment of specific peptides withquantitative measurement of those peptides by mass spectrometry. Inorder to detect and quantitatively measure protein analytes, the SISCAPAtechnology makes use of anti-peptide antibodies (or any other bindingentity that can reversibly bind a specific peptide sequence of about5-20 residues) to capture specific peptides from a mixture of peptides,such as that arising, for example, from the specific cleavage of aprotein mixture (like human serum or a tissue lysate) by a proteolyticenzyme such as trypsin or a chemical reagent such as cyanogen bromide.By capturing a specific peptide through binding to an antibody (theantibody being typically coupled to a solid support either before orafter peptide binding), followed by washing of the antibody:peptidecomplex to remove unbound peptides, and finally elution of the boundpeptide into a small volume (typically achieved by an acid solution suchas 5% acetic acid), the SISCAPA technology makes it possible to enrichspecific peptides that may be present at low concentrations in the wholedigest, and therefore undetectable in simple mass spectrometry (MS) orliquid chromatography-MS (LC/MS) systems against the background of moreabundant peptides present in the mixture. It also provides a sample thatis less complex, and therefore exhibits lesser ‘matrix effects’ andfewer analytical interferences, than observed in the starting digest.This in turn permits mass spectrometry analysis without furtherseparation steps, although additional separation processes could be usedif desired. The sample can be concentrated prior to analysis ifnecessary, but this concentration does not provide any further analytepeptide separation. This enrichment step is intended to capture peptidesof high, medium or low abundance and present them for MS analysis: ittherefore discards information as to the relative abundance of a peptidein the starting mixture in order to boost detection sensitivity. Thisabundance information, which is of great value in the field ofproteomics, can be recovered, however, through the use of isotopedilution methods: the SISCAPA technology makes use of such methods(e.g., by using stable isotope labeled versions of target peptides) incombination with specific peptide enrichment, to provide a method forquantitative analysis of peptides, including low-abundance peptides.

The approach to standardization in SISCAPA is to create a version of thepeptide to be measured which incorporates one or more stable isotopes ofmass different from the predominant natural isotope, thus forming alabeled peptide variant that is chemically identical (ornearly-identical) to the natural peptide present in the mixture, butwhich is nevertheless distinguishable by a mass spectrometer because ofits altered peptide mass due to the isotopic label(s). In oneembodiment, the method for creating the labeled peptide is chemicalsynthesis, wherein a peptide with chemical structure identical to thenatural analyte can be made while incorporating amino acid precursorsthat contain heavy isotopes of hydrogen, carbon, oxygen or nitrogen(e.g., ²H, ¹³C, ¹⁸O or ¹⁵N) to introduce the isotopic label. In theoryone could also use radioactive (i.e., unstable) isotopes (such as ³H),but this is less attractive for safety reasons. The isotopic peptidevariant (a Stable Isotope-labeled Standard, or SIS) is used as aninternal standard and is added to the sample peptide mixture at a knownconcentration before enrichment by antibody capture. The antibodycaptures and enriches both the natural and the labeled peptide together(having no differential affinity for either) according to their relativeabundances in the sample. Since the labeled peptide is added at a knownconcentration, the ratio between the amounts of the natural and labeledforms detected by the final MS analysis allows the concentration of thenatural peptide in the sample mixture to be calculated. Thus, theSISCAPA technology makes it possible to measure the quantity of apeptide of low abundance in a complex mixture, and since the peptide istypically produced by quantitative (complete) cleavage of sampleproteins, the abundance of the parent protein in the mixture of proteinscan be deduced. The SISCAPA technology can be multiplexed to covermultiple peptides measured in parallel, and can be automated throughcomputer control to afford a general system for protein measurement.Creating a new protein-specific assay thus, requires only that apeptide-specific antibody and a labeled peptide analog be created. Afeature of the SISCAPA technology is directed at establishingquantitative assays for specific proteins selected a priori, rather thanat the problem of comparing all of the unknown components of two or moresamples to one another. It is this focus on specific assays that makesit attractive to generate specific antibodies to each monitor peptide(in principle one antibody binding one peptide for each assay). Prior tothe development of SISCAPA it was believed that anti-peptide antibodieswere neither sufficiently specific nor had sufficient affinity to allowisolation of a single peptide from a complex digest. Previouslydescribed methods have not focused on anti-peptide antibodies, but usedinstead general affinity concepts that would bind and enrich all of aclass of peptides by recognizing a ligand, label or feature common tothe class: e.g., immobilized metal affinity chromatography (IMAC) toselect phosphopeptides as a group, anti-phosphotyrosine antibodies toselect phosphotyrosine-containing peptides as a group, or lectins toselect glycopeptides as a group. The SISCAPA technology provides a meansto enrich each peptide sequence specifically with a different antibody(or other equivalently selective binding reagent).

An alternative to using SIS peptides is to use multiple copies of SISpeptides arranged as a linear polypeptide strand known as polySISpeptides. PolySIS peptides have been described, for example, in U.S.patent application Ser. No. 11/147,397 and may be prepared chemically,in vitro or in vivo using the same techniques used for SIS peptides.PolySIS peptides may also be prepared in “extended SIS” form and coupledto a carrier in the same fashion that SIS peptides or extended SISpeptides are attached. In one embodiment polySIS polypeptides comprise arepeat of a single peptide that may be used as a monitor peptide. Inanother embodiment, polySIS polypeptides (which may also be termedpolySIS proteins) have an amino acid sequence containing several (atleast two, three, four, five or more) amino acid subsequences found innature and wherein at least two different subsequences act as monitorsequences. In such an embodiment, the subsequences of the polySISpolypeptide are part of at least one (or alternatively two, three, four,five or more) natural proteins that is/are target protein(s) whoselevels will be assessed. The subsequences of polySIS peptides contain attheir ends cleavage sites that can be cleaved by the same site-specificproteolytic treatment to release said subsequences from the nativeproteins in the test sample. Thus, polySIS peptides may be used as aninternal standard in the same fashion as SIS peptides and also as asingle internal standard for multiple protein analytes, wherein theratio of the monitor peptide sequences will be fixed by the number oftimes the subsequence appears in the polySIS peptide.

A further objective of SISCAPA is to deliver a series of differentmonitor peptides (selected by a corresponding series of specificantibodies and potentially derived by digestion of different sampleproteins) to a mass spectrometer at very nearly the same abundance(e.g., within a factor of 500, 250, 200, 100, 50, 20, or 10 fold molarabundance) and free of other extraneous peptides. By equalizing theabundance of a series of peptides through antibody capture of aselectable fraction of each peptide, the method can ensure that all thecaptured peptides are present within the mass spectrometer's dynamicrange and that this dynamic range can be optimally employed in spanningthe true dynamic range of the peptide analytes. If the MS system has adynamic range of 1000 (a range of 100 to 10,000 is typical depending onthe type of MS), the method attempts to ensure that the peptides ofinterest are presented to the MS at a level in the middle of that range,thus, allowing an optimal capacity to detect increases or decreases inrelative abundance of the natural and isotopically labeled forms. If thepeptides were presented to the MS at different abundances (e.g., thosepeptides at relative concentrations of 1, 0.001 and 1,000), then the MSwill have great difficulty in detecting equivalent quantitativedifferences between natural and isotopically labeled forms of thesethree peptides. By “flattening” the abundance distribution of thepeptides the quantitative resolution is substantially enhanced.

The basic SISCAPA embodiment combines 1) methods for creation andaffinity purification of antibodies that tightly but reversibly bindshort peptide sequences; 2) methods for digestion of complex proteinmixtures to yield short peptides; 3) methods for synthesis of definedpeptides containing isotopic labels; 4) methods for efficient captureand release of peptides; and 5) methods for MS measurement of ratios oflabeled and unlabeled (sample-derived) peptides to yield a quantitativemeasurement.

In the simplest SISCAPA embodiment, the following steps are carried outfor each protein to be measured in plasma (or another sample containingprotein(s)) in order to generate a specific quantitative assay system.The starting point is protein identification, which typically is basedon the sequence of known proteins identified by accession number in asequence database such as SwissProt or Genbank. The steps include one ormore of:

Selecting a Monitor Peptide (Step a)

Using the known sequence of the protein, one or more peptide segmentswithin the protein are selected as “monitor peptides.” A good monitorpeptide satisfies a set of criteria designed to select peptides that canbe chemically synthesized with high yield, that can be detectedquantitatively in an appropriate mass spectrometer, that arereproducibly cleaved from the parent protein during digestion, and thatelicit antibodies when used as antigens, although any peptide resultingfrom cleavage with the desired enzyme is a possible choice. One usefulset of criteria is the following:

i: The peptide has a sequence that results from cleavage of the proteinwith a desired proteolytic enzyme (e.g., trypsin). All the candidatetryptic peptides can be easily computed from the protein sequence byapplication of generally available software.

ii: The peptide should be hydrophilic overall, and soluble inconventional solvents used in enzymatic digestion and affinitychromatography. Hydrophilic peptides can be selected based on computedscores obtained for each peptide from generally available softwareprograms. In general the hydrophilic peptides are those that containmore polar amino acids (his, lys, arg, glu, asp) and fewer hydrophobicamino acids (trp, phe, val, leu, ile).

iii: The peptide should contain a) no cys, because a c-terminal cys maybe added for convenience in conjugation of the immunogen (the presenceof two cys in a peptide can lead to undesirable dimerization andcross-linking) and because the alkylation of peptide cysteines can beirreproducible; and b) no met, because of the possibility of oxidationof this amino acid.

iv: The peptide should ionize well by either electrospray (ESI) ormatrix-assisted laser desorption (MALDI) ionization. This characteristiccan be estimated by software programs or determined experimentally by MSanalysis of a digest of the protein in question to see which peptidesare detected at highest relative abundance.

v: The peptide should be immunogenic in the species in which theantibody will be raised. Immunogenicity is generally better for peptidesthat are hydrophilic (compatible with (ii) above); that include a bendpredicted by secondary structure prediction software; that include noglycosylation sites; and that are 10-20 amino acids in length.

vi: The peptide should not include within it the sites of any commonsequence polymorphisms (i.e., genetic variants) in the target protein(as this could affect the estimation of the respective protein'sabundance if the variant peptide does not appear at the expected mass).

vii: the peptide should not share appreciable homology with any otherprotein of the target organism (as determined for example by the BLASTsequence comparison program). This characteristic should tend to reduceany interference in the antibody capture step from peptides originatingin proteins other than the target.

viii: the peptide should be produced at reproducible (ideally high,close to 100%) stoichiometric yield by tryptic (or other) digestion ofthe starting sample.

ix: the peptide and its fragments measured by mass spectrometry (e.g.,by selected or multiple reaction monitoring using a triple quadrupoleMS) should produce comparable results at multiple analysis sites and ondifferent mass spectrometers (i.e. yielding good site to sitereproducibility).

All possible peptides derived from the target protein can be evaluatedaccording to these criteria, either through computation or experimentalanalysis of digests of isolated target proteins or biological samplesincluding the target protein, and one or more peptides selected thatbest balance the requirements of the method. A database of all thepeptides (e.g., tryptic peptides) and their derived properties for afinite set of analytes such as the known proteins in plasma can becreated and used as a basis for selection of monitor peptides. Beginningwith the known amino acid sequences of protein analytes, efficientalgorithms can construct all the possible tryptic peptides that will becreated by trypsin digestion of the protein. These tryptic peptidesequences can be stored as records in a database, and similar recordsgenerated for other possible cleavage enzymes and reagents. Additionalalgorithms can be employed to compute various physical and biologicalproperties of each peptide, including length, mass, net charge atneutral pH, propensity to adopt secondary structure, hydrophilicity,etc. These derived data can be tabulated for each peptide, andadditional aggregate calculations performed to develop prioritizingscores associated with likelihood of success as a monitor peptide. Thesepriority scores can be sorted to select candidate monitor peptides foreach protein.

Creating Isotope-Labeled Monitor Peptides (Step b)

An isotopically labeled version of the selected peptide (a “SIS”) isthen made in which the chemical structure is maintained, but one or moreatoms are substituted with an isotope such that an MS can distinguishthe labeled peptide from the normal peptide (containing the naturalabundance of each element's isotopes). For example, nitrogen-15 orcarbon-13 could be introduced instead of the natural nitrogen-14 orcarbon-12 at one or more positions in the synthesized peptide. Thesynthesized peptide will be heavier by a number of atomic mass unitsequal to the number of substituted nitrogens or carbons. The peptide iscarefully made so that the number of added mass units is known andwell-determined (i.e., all of the material produced as one standard hasthe same mass insofar as possible—achieved by using highly enrichedisotopic variants of the amino acids, for example). In one embodiment,nitrogen-15 or carbon-13 labeled amino acid precursors substitutedat >98% are used at one or more positions in the peptide synthesisprocess to introduce between 4 and 10 additional mass units compared tothe natural peptide. Such nitrogen-15 labeled amino acid precursors (ortheir carbon-13 labeled equivalents) are commercially available as FMOCderivatives suitable for use directly in conventional commercial peptidesynthesis machines. The resulting labeled monitor peptides can bepurified using conventional LC methods (typically to >90% purity) andcharacterized by MS to ensure the correct sequence and mass.

Creating Anti-Peptide Antibodies (Step c)

To immunize an animal for production of anti-peptide antibodies, thesame peptide (labeled or not, if the latter is, as expected, moreeconomical) is coupled to a carrier protein (e.g., keyhole limpethemocyanine (KLH); not homologous to a human protein) and used toimmunize an animal (such as a rabbit, chicken, goat or sheep) by one ofthe known protocols that efficiently generate anti-peptide antibodies.For convenience, the peptide used for immunization and antibodypurification may contain additional c-terminal residues added to themonitor peptide sequence (here abbreviated MONITOR), e.g.:nterm-MONITOR-lys-gly-ser-gly-cys-cterm (SEQ ID No. 10). The resultingextended monitor peptide can be conveniently coupled to carrier (e.g.,KLH) that has been previously reacted with a heterobifunctional reagentsuch that multiple SH-reactive groups are attached to the carrier. Inclassical immunization with the peptide (now as a hapten on the carrierprotein), a polyclonal antiserum will be produced containing antibodiesdirected to the peptide, to the carrier, and to other non-specificepitopes. Alternatively, there are many methods known in the art forcoupling a peptide, with or without any extensions or modifications, toa carrier for antibody production, and any of these may be used.Likewise there are known methods for producing anti-peptide antibodiesby means other than immunizing an animal with the peptide on a carrier.Any of the alternatives can be used provided that a suitable specificreversible binding agent for the monitor peptide is produced.

Specific anti-peptide antibodies (e.g., rabbit antibodies such as rabbitmonoclonal antibodies) are then prepared from this antiserum by affinitypurification on a column containing tightly-bound peptide. Such a columncan be easily prepared by reacting an aliquot of the extended monitorpeptide with a thiol-reactive solid support. Crude antiserum can beapplied to this column, which is then washed and finally exposed to 10%acetic acid (or other elution buffer of low pH, high pH, or highchaotrope concentration) to specifically elute antipeptide antibodies.These antibodies are neutralized or separated from the elution buffer(to prevent denaturation), and the column is recycled to physiologicalconditions for application of more antiserum if needed. Antibodies alsomay be produced by hybridoma techniques well know in the art.

The peptide-specific antibody is finally captured or immobilized on acolumn, bead or other surface for use as a peptide-specific affinitycapture reagent. In one embodiment, the anti-peptide antibody isimmobilized on commercially available protein A-derivatized POROSchromatography media (Applied Biosystems) and covalently fixed on thissupport by covalent crosslinking with dimethyl pimelimidate according tothe manufacturer's instructions. The resulting solid phase media canbind the monitor peptide specifically from a peptide mixture (e.g., atryptic digest of serum or plasma) and, following a wash step, releasethe monitor peptide under mild elution conditions (e.g., 10% aceticacid). Restoring the column to neutral pH then regenerates the columnfor use again on another sample, a process that is well known in the artto be repeatable hundreds of times. In another embodiment, theanti-peptide antibodies are captured on magnetic beads (either beforeexposure to the digest or afterwards), which simplifies separation ofantibody from the digest after peptide binding, washing, and peptideanalyte recovery. High affinity (typical dissociation constants of 10⁻⁹to 10⁻¹¹), high specificity antibodies are preferred, and processes ofantibody generation and selection are designed to optimize thesecharacteristics.

Digestion of Sample to Peptides (Step d)

A protein sample such as plasma, containing the selected protein to bemeasured, is digested essentially to completion with an appropriateprotease (e.g., trypsin) to yield peptides (including the monitorpeptide selected in step 1). For a monitor peptide whose sequenceappears once in the target protein sequence, this digestion generatesthe same number of monitor peptide molecules as there were targetprotein molecules in the stating sample (provided each monitor peptidesequence occurs once per protein). The digestion is carried out by firstdenaturing the protein sample (e.g., with urea, trifluoroethanol orguanidine HCl), reducing the disulfide bonds in the proteins (e.g., withdithiothreitol or mercaptoethanol), alkylating the cysteines (e.g., byaddition of iodoacetamide), quenching excess iodoacetamide by additionof more dithiothreitol or mercaptoethanol, and finally (after removal ordilution of the denaturant) addition of the selected proteolytic enzyme(e.g. trypsin), followed by incubation to allow digestion. Followingincubation, the action of trypsin is terminated, either by addition ofan enzyme inhibitor (e.g., DFP, PMSF or aprotinin) or by denaturation(through heat or addition of denaturants, or both) or removal (if thetrypsin is on a solid support) of the trypsin. The destruction of thetrypsin activity is important in order to avoid damage to antibodieslater by residual proteolytic activity in the sample.

Adding Isotopically-Labeled Monitor Peptide Internal Standards (Step e)

A measured aliquot of isotopically-labeled synthetic monitor peptide(SIS) is added to a measured aliquot of the digested sample peptidemixture in a known amount, typically close to or greater than (if thestandard serve for example, as carrier for a low abundance peptide) theexpected abundance of the same “natural” peptide in the sample aliquot.Following this addition the monitor peptide will be present in thesample in two forms (natural and isotopically-labeled). Theconcentration of the isotopically-labeled version is accurately knownbased on the amount added and the known aliquot volumes. The labeledpeptide may be added separately from the antibody, although it can beadded in combination with the antibody for stability and simplicity.

Enrichment of the Monitor Peptide by Antibody Capture and Elution (Stepf)

The peptide mixture (sample digest with added isotopically-labeledmonitor peptides) is exposed to the peptide-specific affinity capturereagent, which preferentially binds the monitor peptide but does notdistinguish between labeled and unlabeled forms (since isotopicsubstitutions are not expected to affect antibody binding affinity).Following one or more wash steps (e.g., phosphate-buffered saline,water) the bound peptides are then eluted (e.g., with 5% acetic acid, orwith a mixture of water, formic acid and acetonitrile), for MS analysis.The affinity support can, if desired, be recycled in preparation foranother sample. In some of the high-throughput assay applicationsenvisioned, it will be advantageous to recycle the immobilizedantibodies hundreds, if not thousands, of times when flow-throughcolumns are used. Current evidence indicates that rabbit polyclonalantibodies can be recycled at least 200 times when antigens are elutedwith 5% or 10% acetic acid and total exposure to acid is kept short(e.g., less than 1 minute before regeneration with neutral pH buffer).In a capillary column format, where the immobilized antibody bed can besubmicroliter in size, the duration of acid exposure could be furtherdecreased, possibly extending the life of the immobilized antibodyadsorbent even further. Magnetic bead embodiments can also beconveniently automated in a variety of ways to achieve high throughput,and avoid the potential for carryover inherent in re-usable antibodycolumns.

The enrichment step is an important element of the method because itallows enrichment and concentration of low abundance peptides, derivedfrom low abundance proteins in the sample. Ideally, this enrichmentprocess delivers only the monitor peptide to the MS, and makes itsdetection a matter of absolute MS sensitivity, rather than a matter ofdetecting the monitor peptide against a background of many other,potentially much higher abundance peptides present in the whole sampledigest. This approach effectively extends the detection sensitivity anddynamic range of the MS detector in the presence of other high abundanceproteins and peptides in the sample and its digest.

Analysis of the Captured Monitor Peptides by Ms (Step g).

The monitor peptide (including natural and isotopically-labeledversions) enriched in the preceding step is delivered into the inlet ofa mass spectrometer (e.g., by MALDI or by electrospray ionization(ESI)). The mass spectrometer can be a TOF (time-of-flight), a Q-TOF, aTOF/TOF, a triple quadrupole, an ion trap, an orbitrap, an ion-cyclotronresonance machine, or any other instrument of suitable mass resolution(>1,000) and sensitivity, and can employ one, two, or more levels ofmass selection interspersed with analyte fragmentation processes (e.g.,collision-induced fragmentation).

The MS measures the ion current or ion count (number of ions) for bothversions of the monitor peptide (natural and labeled), typically as afunction of time or within an accumulated spectrum (in the case ofTOF-MS). The ion current may be integrated over time (ideally for aslong as the monitor peptide appears in the mass spectrum) for each massspecies, and the integrated amounts of natural and isotope-labeled formsare computed as measures of peptide amount. Alternatively the maximumpeak heights of the natural and labeled peptides can be used as measuresof peptide amount.

Computation of Abundance of Each Monitor Peptide in the Sample (Step h)

A ratio is computed between the amounts of the labeled and unlabeled(natural) monitor peptides. Since the amount of labeled peptide added isknown, the amount of the natural monitor peptide derived from the sampledigest can then be calculated by multiplying the known concentration oflabeled monitor peptide by this measured ratio. By assuming that theamount of the monitor peptide in the digest is the same as (or closelyrelated to) the amount of the parent protein from which it is derived, ameasure of the protein amount in the sample can be obtained.

The foregoing description outlines the SISCAPA method, which while oneembodiment of the application of the present technology, is not the onlyapplication envisioned.

2 Determination of Clinical Cutoff and Clinical Reference Interval.

-   -   For quantitative tests in which deviation from ‘normal’ in one        direction is significant (e.g., a level of a cancer marker like        PSA or CAl25 above a cutoff value) but where deviation in the        other direction (usually lower than ‘normal’) is not considered        significant, only one threshold—the test “cutoff”—is needed. For        tests in which a deviation from a normal range in either        direction can be significant, test values are typically        interpreted against the upper and the lower bounds of the        established reference range. Physicians frequently use this        method of interpretation to identify significant test results        that are likely to bear on a patient's diagnosis or treatment.        Cutoffs and reference range boundaries are typically established        by making analyte measurements in a large group of patients,        developing the histogram of these values and selecting points        for which only a small fraction (usually ˜2.5%) of the ‘normal’        population give results outside the points. Cutoffs or reference        intervals can be established as part of FDA approval processes,        and may be superseded by reference range studies within a        clinical institution to better reflect the normal population of        patients in that institution, or even by personal reference        values used in interpreting individual patient test results over        time.

3 Internal Standard Peptides and Proteins, and Isotopic LabelingSchemes.

-   -   SIS peptides can be synthesized containing a variety of        combinations of labeled amino acids. Thus, different versions of        the same SIS peptide can be made that, while chemically        identical to the analyte peptide, are nevertheless        distinguishable from the analyte and from each other by mass.        Such different peptides can be used together in an assay to        provide two or more internal standards spiked into the sample or        its digest at different levels.

4 Providing Internal Standards at Clinical Cutoff and Reference Levels.

-   -   The level(s) of one or more internal standards can be selected        to equal or approximate clinically important analyte        concentrations. Using standards present at clinical decision        levels has major advantages in an analytical procedure. First,        it is it possible to confirm the result of the test by        inspection of raw MS data: in almost all cases it is clear        whether the analyte peptide produces a signal that is greater        than or less than that of the analogous internal standard. This        is true whether peak height or a peak area is compared.        Secondly, the precision of comparing the amount of an analyte to        an internal standard is typically highest when the two are        present at near equal amounts in a standardized sample: this        minimizes the effect of any non-linearity in the response of the        MS detector (which may become significant when analyte and        standard are present in different amounts), and eliminates any        uncertainty as to the true position of the decision value in        relation to the internal standard. Thirdly, when analyte and        standard are present at near-equal amounts, the signal-to-noise        and statistical precision of the two measurements are likely to        be very similar, thereby simplifying the quality control of an        assay. Thus, if the assay yields a measurement for an internal        standard that is large enough to ensure adequate precision        (e.g., in comparison with prior quality studies relating        magnitude of such measurements with replicate precision) then a        single measurement of analyte in a sample yielding a similar        amount is likely to have similarly adequate precision. Finally,        “hard-wiring” a decision threshold into an assay kit, for        example by depositing an amount of SIS in or on a single-use        component of an assay kit such that the provided amount of SIS        dissolves in an accurately measured amount of sample, provides a        major advance in the reliability of the overall assay result.

5 Comparison of Analyte Levels to Internal Standards: MALDI and ESI.

-   -   The present technology provides an improved method for assessing        analyte concentration in the sample against the appropriate        clinical criteria (either a cutoff or a reference range). The        principal is illustrated for the case of MALDI-TOF mass        spectrometry in FIG. 1A, where the peak height for the major        isotopic form of internal standard 1 establishes the cutoff        level 3 equal to the assay cutoff: the concentration of the        standard is set deliberately to equal this clinically determined        cutoff. The measured height of analyte peak 2 is compared to        cutoff 3 and easily judged to be less than the cutoff, thus        providing the result of the binary test. In FIG. 1B, the        alternative situation is shown, in which the analyte peak 2 is        taller than the cutoff 3 set by the internal standard 2,        indicating that the analyte concentration exceeds the clinical        threshold. It is equally possible, though less intuitive and        direct, to use alternative measures such as peak area in place        of peak height. The advantage of peak height in this context is        that the result can be ‘read’ directly from the primary MS        output—a plot of signal intensity versus either mass (or m/z) in        TOF-MS, or versus time in liquid chromatography mass        spectrometry methods (LC-MS). The mass accuracy of modern TOF        mass analyzers is sufficient (1-20 ppm) to provide strong        evidence of the identity of the expected analyte and SIS        peptides. FIG. 2 illustrates the comparison of the analyte (peak        2) with SIS internal standard 1, which establishes the low end 3        of the reference interval, and with a different SIS internal        standard 4, which establishes the upper end 5 of the reference        interval. It is immediately evident that the analyte        concentration lies within the reference interval. FIG. 3 shows        the equivalent situations measured by selected reaction        monitoring LC-MS/MS, carried out using a triple quadrupole MS.        In this case sample peptides are separated over time by        reversed-phase liquid chromatography, but, being chemically        identical, the analyte and SIS internal standards elute        simultaneously. In FIG. 3A, analyte peak 8 has both less height        and lower peak area than internal standard peak 7 indicating a        test result of less than the cutoff 3; in FIG. 3B, the        alternative situation (analyte amount greater than the cutoff)        is shown. FIG. 4 shows the LC-MS cases in which analyte peak 8        is below the reference interval lower bound 3 (FIG. 4A); between        reference interval boundaries 3 and 5 (FIG. 4B); or above the        reference interval upper bound 5 (FIG. 4C). As in the case for        MALDI peaks, the result of the test is immediately evident        through comparison of the analyte peak with internal standards        set at the clinical cutoff or reference interval upper and lower        bounds. When desirable, as for example in the context of        personalized medicine, the cutoff or reference interval can be        set based on a patient's past analytical history (the past        values of tests for the same analyte). This approach takes into        account the differences between individuals by using the        individual as his or her own control. It is more difficult        technically to provide individually personalized internal        standard concentrations in routine assays, but this can be done        using a database of personal test values and accurate technology        for spiking variable levels of standards in each sample        analyzed. Thus, in one embodiment the amount of internal        standard utilized will be an amount based upon past measurements        of an individual patient (subject).

When peak heights and peak areas are obtained within a spectrum ofsufficient resolution so as to effectively resolve isotopic peaks, suchas when conducting comparisons of peak heights and peak areas obtainedwithin a spectrum (e.g., using MALDI-TOF mass spectrometry), one or morespecific isotopic composition peaks from the natural and labeled formscan be selected for comparison. With such resolved isotopic peaks it isfacile to compare peak heights directly in a raw or smoothed spectrum,and this provides a preferred means of determining the relationshipbetween an analyte and a corresponding SIS present at a test evaluationthreshold so as to deliver a test result. Peak areas can be similarlycompared after computation by summing measurements within the peak(e.g., by numerical integration) or by fitting an empirical or modeledpeak shape to the spectral data to obtain the peak area.

When peaks occur in a time domain (e.g., MRM peaks observed during thecourse of a chromatographic separation of peptides), such as whenconducting comparisons of peak heights and peak areas using LC-MS/MS,additional instrument factors may come into play. In a triple quadrupolemass spectrometer, resolution is typically not sufficient to deliverbaseline separation between isotopic forms, and the mass-domain envelopedetermining what is detected may not be a rectangular step function, butanother peak distribution, the width of which can be altered byinstrument settings (e.g., the “resolution” setting common on triplequadrupoles). Because of these features, the height or area obtained fora specific isotopic peak may vary slightly depending on the relativeabundances of adjacent isotopic forms of a peptide, varying amounts ofwhich may be enclosed within the detected envelope along with the targetisotopic form. These relative abundances often differ between labeledand unlabeled peptides, in large part because of the use of heavy labelisotopes with less than perfect levels of enrichment (¹³C and ¹⁵N areoften available at 98% isotopic substitution, but not 100%), resultingin different relative abundances of peaks adjacent to the labeled vsunlabeled peaks to be measured. Since different specific instrumentdesigns and tuning parameters may therefore alter slightly the observedratios between analyte and SIS peaks, it may be necessary to apply acorrection factor to the measured ratio to obtain an accurate comparisonbetween the two. Such a factor can be determined accurately andprecisely from calibration experiments using synthetic peptides. Whenthe present invention makes use of peak height and/or area comparisons,the use of such correction factors is optionally incorporated in thedata processing method used.

6 Precision and Accuracy of MS Measurements.

-   -   Given the fact that MS measurements are not infinitely precise,        occasions will arise in which a test analyte occurs at a level        very close to the SIS internal standard level. In the event that        the analyte level occurs within a defined interval around the        SIS level, for example a statistically defined interval such as        +/−3, 2, 1, ½ or ¼ standard deviations determined from the SIS        measurement, then it may be advantageous to report a ‘grey area”        or indeterminate result. An advantage of the present approach is        that such a grey area interval can be extremely narrow, and        hence occur in an extremely small subset of samples.

7 Multiplex Tests Measuring Multiple Analytes.

-   -   Multiplex tests measuring multiple analytes can be evaluated by        comparison with multiplex SIS peptides present at pre-determined        test evaluation thresholds. In the simplest case, the amount        (e.g., peak height) of each analyte is compared to the amount of        the corresponding SIS (present at an analyte-specific decision        level) to obtain a binary (“High” or “Low”) result. These binary        results are then evaluated against a matrix of possibilities (in        principle all possible result patterns), each of which is        associated uniquely with an overall test result, and the result        corresponding to the matching pattern is delivered as the        result.

8 Packaging Peptides on a Carrier to Avoid Loss.

-   -   Providing SIS peptides in assays at concentrations accurately        representing clinical decision, or other, values or ranges        requires accurate and precise control of peptide delivery, and        consequent minimization of peptide loss or deterioration prior        to use. Synthetic peptides of the size and composition of        typical tryptic (or other cleavage agent-produced) peptides        exhibit a wide range of physical properties, particularly as        regards solubility, and conversely, stickiness to polymer,        glass, and other surfaces found in storage containers,        laboratory ware, pipettes, LC tubing and the like. Peptides with        sticky character, often associated with high hydrophobicity,        frequently exhibit poor recovery from storage containers or        during handling processes. Recovery is typically worst for        peptides at low abundance (when the ratio between surface        adsorptive sites and peptide is high), and it is well known that        recovery of femtomole amounts of peptides from typical        polypropylene storage tubes can be very poor. In one embodiment,        the technology described herein formulates peptides in such a        way that such losses are minimized, thus providing improved        recovery of peptides for use, e.g., as internal standards in        mass spectrometric assays such as the SISCAPA method. One        approach described here to improve recovery is to link peptides        to a larger carrier molecule or particle having good storage and        recovery properties, e.g., a large hydrophilic, soluble protein        or else a visible particulate bead (e.g., a magnetic bead). In        suitably designed carriers, the bulk properties of the carrier        override the problematical properties of the peptide(s), giving        the combined object the desired high recovery from storage.

9 Linkage Between a Peptide and a Carrier.

-   -   A variety of linkage interactions are possible for coupling        peptides to a carrier, including covalent reactions between        sulfhydryls (e.g., cysteine) and maleimides, “Solulink        chemistry” (reaction of 6-hydrazino-nicotinamide (HyNic) with        carbonyl moieties), etc., or non-covalent but tight interactions        such as those between biotin and avidin (or streptavidin), and        between a hexa-histidine tag and immobilized nickel. In one        embodiment, the peptide linkage site consists of a cysteine        sulfhydryl, the C residue comprising a part of the extension        sequence of an extended SIS peptide, and the carrier linkage        site consists of a maleimide group covalently attached to a        protein carrier such as KLH. Alternatively, a peptide may be        provided bound non-covalently to a surface that contacts the        sample or digest at some point in the analytical workflow, such        that the peptide is released by dissolution upon such contact.        In other embodiments, the polySIS peptides are immobilized on a        carrier using any of the above-described linking interactions.

10 Recovery of Peptides from a Carrier by Proteolytic Cleavage.

-   -   For many of the applications for synthetic peptides described        here, particularly use as internal standards in analytical mass        spectrometry, the SIS peptide material must ultimately be        delivered as a molecule of the correct length (i.e., the same        length and structure as the analyte for which it serves as an        internal standard) free in solution at a known concentration.        Thus, it is desirable for all (or at least some known fraction)        of the peptide(s) packaged on a carrier to be released and made        available as an internal standard for direct MS quantitation of        the corresponding sample-derived target peptide or else for        treatment by some selection, fractionation or specific capture        method that enriches the analyte peptide and the SIS together,        preserving their ratio to provide analyte concentration upon MS        analysis. Proteolytic cleavage of a sample into peptides is a        common feature of sample preparation for analysis of protein        samples by MS, including those involving a specific enrichment        step (e.g., the SISCAPA method). It is possible and efficient        therefore to use the same proteolytic process to liberate        peptides from a carrier, and in the simplest case to accomplish        this at the same time as sample digestion by adding the        peptide-carrier conjugate to the sample before digestion,        thereafter digesting both sample and conjugate together. In        particular when the peptide linkage site connecting the peptide        to the carrier is present in the extension portion of the        extended peptide (i.e., the portion not within the SIS peptide)        then proteolytic cleavage of the bond between the SIS and the        extension will release intact SIS from the carrier (with the        peptide linkage site and the extension remaining on the        carrier). In the case where the proteolytic cleavage is carried        out by the enzyme trypsin, then the junction between SIS        sequence and the extension is a tryptic cleavage site (i.e., a K        or R residue followed by any amino acid except P). If the        extension portion of the peptide is n-terminal to the SIS        sequence, then the extension will consist of an arbitrary        sequence of amino acids ending in K or R, and the tryptic        cleavage will occur after this K or R, releasing the c-terminal        SIS sequence. If the extension is c-terminal to the SIS        sequence, then, since the SIS (having the same sequence as a        tryptic peptide) will end in K or R the SIS sequence will be        cleaved by trypsin from the c-terminal extension sequence,        provided that the extension sequence does not begin with a        proline. Extension sequences can contain additional amino acid        residues to space the cleavage site away from the carrier,        reducing steric hindrance to the action of the cleavage enzyme.        Extension sequences can be generic (such as GSG or GSGSG        sequences (SEQ ID Nos. 1 and 2, respectively) or they can        comprise flanking sequence from the target protein sequence        (i.e., an n-terminal extension could consist of a stretch of        amino acid sequence immediately n-terminal to the analyte        peptide sequence). The latter approach provides a cleavage site        sequence environment most closely approximating that in the        sample protein target, and therefore may help to normalize SIS        release to parallel release of analyte peptide during sample        digestion.

11 Surrogate Quantitation of SIS Peptides by Measuring the Amount ofCarrier.

-   -   An easily quantitated or identified physical or chemical        property of the carrier can allow facile detection and        measurement of its amount. Suitable properties include optical        absorbance (e.g., for naturally colored protein molecules such        as the heme-binding domain of cytochrome b5), fluorescence        (e.g., found in the green fluorescent protein and its        derivatives), content of biotin groups (which can be assayed        competitively or directly using well-known biotin binding        proteins such as avidin or streptavidin), magnetic properties        (e.g., measurable in magnetic beads or the protein ferritin),        weight (e.g., dry mass), or even countability (e.g., for 2-50        micron diameter bead materials which can be counted directly in        a flow cytometer). In some cases two properties can be used        together to establish an improved quantitation method, such as        when particles carrying a fluorescent chromophore are measured        in a flow cytometer that is capable of both quantitating        fluorescence of each particle and counting the particles. Using        these or similar properties, the amount of carrier can be        directly determined at any time, from manufacture until use in        an assay. Provided that a quantitative determination is made of        the amount (e.g., moles) of peptide bound by a measurable amount        of the carrier, then the amount of peptide being added as        carrier conjugate can be determined indirectly by quantitation        of the carrier at any subsequent time. This approach makes it        possible to know and specify the amount of added carrier and        attached SIS peptide at the time of addition to the sample        (before or after digestion), and can be used both to verify        addition of the desired amount of SIS internal standard, and to        control addition to ensure the correct amount is added (e.g.,        when the added carrierSIS material passes a quantitative sensor        that provides feedback to a valve such that the valve opens to        allow a predetermined measured quantity of the carrierSIS to        pass by, after which the valve shuts.)

12 Use of a Magnetically Manipulatable Carrier.

-   -   A carrier consisting of magnetic beads is particularly favorable        due to the ease with which it can be transported into reactions        with minimal added fluid volume (thus avoiding dilution), and        the ease with which the carrier can be removed from a sample        following the release of peptide (e.g., by proteolytic        cleavage). Such beads are available in a range of sizes from 200        microns to 20 microns to 1 micron to less than 100 nm diameter,        and with a variety of surface coatings. Extended SIS peptides        having an n-terminal CGSG-extension (SEQ ID No. 3) can be        coupled to beads that have been previously coated with a protein        activated with 4-(N-Maleimidomethyl)cyclohexane-1-carboxylic        acid 3-sulfo-N-hydroxysuccinimide ester (a heterobifunctional        crosslinking reagent that reacts with protein primary amino        groups to provide sulfhydryl-reactive maleimide groups on the        protein surface). Immobilization of the protein on a magnetic        bead surface facilitates carrying out this stepwise treatment,        in which beads can easily be moved from reagent to reagent, as        well as final washing of the bead-SIS peptide conjugate to        remove any weakly or non-specifically bound peptide. Numerous        alternative methods of linking peptides to beads of various        forms are known to those skilled in the art.

13 Packaging Collections of Peptides at Defined Stoichiometry onCarriers.

-   -   Two or more different peptides, comprising two or more different        SIS peptide sequences, can be linked to the same carrier        preparation in a known stoichiometric relationship (e.g., one        peptide present at 3.5-fold greater molar amount than a second        peptide) yielding a stable mixture whose molar ratio is        protected against perturbation by preferential loss of one        component.

14 Providing Concentration Curves Via Sis on Carriers.

-   -   Two or more different SIS versions of a single analyte peptide        sequence, each differing from the analyte and each other only in        mass, can also be linked to a carrier in established molar        ratios. Such a construct provides a series of distinguishable        peptide internal standards at different known amounts, thus        establishing the basis of an internal calibration curve when        measured quantitatively by mass spectrometry.

15 Providing Mixtures of Carriers.

-   -   Two or more carriers prepared according to these concepts can be        mixed in defined ratios to prepare more complex internal        standards.

Embodiments

1) In a first embodiment, an internal standard stable-isotope labeledpeptide is added to the sample digest prior to antibody enrichment in aSISCAPA assay at a concentration equal to the established testevaluation threshold of the assay. After elution of the capturedpeptides from the SISCAPA capture agent, the peptides are analyzed bymass spectrometry. The result of the test is read by comparing theheight (or area) of the predominant analyte peak with the height (orarea) of the predominant internal standard peak, or more generally bycomparing the height (or area) of one or more pre-specified analytepeaks with the height (or area) one or more pre-specified internalstandard peaks.

2) In another embodiment, first and second internal standardstable-isotope labeled peptides (identical in chemical structure to eachother and to the analyte, but differing in mass) are added to the sampledigest prior to antibody enrichment in a SISCAPA assay at concentrationsequal to i) the established clinical reference interval lower limit, andii) the established clinical reference upper limit, respectively. Afterelution of the captured peptides from the SISCAPA capture agent, thepeptides are analyzed by mass spectrometry (e.g., MALDI massspectrometry). The result of the test is read directly by comparison ofthe height of the predominant analyte peak with the height of thepredominant peaks of each internal standard to determine if the analyteis below, within, or above the reference interval (as illustrated inFIG. 2).

3) In another embodiment, the assay of the previous embodiment iscarried out, but analyzed using selected reaction monitoring MS on atriple quadrupole instrument. The result of the test is read directly bycomparison of the peak area of the predominant analyte peak with thepeak areas of the predominant peaks of each internal standard todetermine if the analyte is below, within, or above the referenceinterval (as illustrated in FIG. 4).

4) In another embodiment, first and second internal standardstable-isotope labeled peptides (identical in chemical structure to eachother and to the analyte, but differing in mass) are added to the sampledigest prior to antibody enrichment in a SISCAPA assay at concentrations(relative to the sample) equal to i) a calculated personal referenceinterval lower limit, and ii) a calculated personal reference upperlimit, respectively. The personal reference interval is computed basedon previous analyses of samples from the same individual, and thusrepresents a personalized reference interval. The addition of SISpeptides is carried out by a computer-controlled dispenser thatdetermines patient identity from the identifying code of the presentsample, uses it to search for previous result values or personalreference values in a database, recovers or computes the most currentand appropriate updated personal reference values, and delivers into thesample amounts of the first and second internal standard peptidesequivalent to the calculated lower and upper limits in relation to theamount of patient sample contained in the present analysis. Afterelution of the captured peptides from the SISCAPA capture agent, thepeptides are analyzed by mass spectrometry to determine if the analytelevel is below, within, or above the reference interval. In all of theabove embodiments, quantitative analyte concentrations can becalculated, as in the original SISCAPA method, from the ratio of analytepeak height or area to SIS peak height or area, reported with the directcomparison test result, and can be stored in a database to enablesubsequent use as a basis of comparison for future test results.

5) In another embodiment, a series of individual analytes is measured ina multiplex assay, and each analyte is compared against its respectiveSIS internal standard which has been added to the sample at itsrespective predetermined multiplex test threshold. Evaluation of themultiplex test result consists by determining a result for eachindividual analyte and then evaluating the panel of analyte resultstogether by lookup in a predetermined outcome table as shown below. Thetable contains multiplex test results for each possible set ofindividual analyte results. A specific test result set, shown on theright, is compared with the table to look up the multiplex result, inthis case positive.

6) In another embodiment, a known molar amount of an extended SISpeptide (corresponding to a selected analyte peptide of a target sampleprotein) is reacted with a molar excess of maleimide groups on activatedKLH carrier protein to form a carrierSIS conjugate. A known amount ofthis conjugate (carrying a known molar amount of extended SIS peptide)is added to a known volume of protein sample such as human plasma, andthe combined spiked sample digested with trypsin to yield trypticpeptides. The analyte peptide (produced by tryptic cleavage of thetarget protein) and the corresponding SIS peptide (produced by cleavagebetween the SIS sequence and the extension linking it to the carrier)are analyzed by quantitative mass spectrometry to provide a ratio ofanalyte:SIS. Multiplying this ratio by the known molar quantity of SISadded as part of the conjugate yields the molar amount of the analytepeptide, and thus provides a measure of the amount of the target proteinin the sample.

7) In another embodiment, a green fluorescent protein (GFP) is used ascarrier instead of KLH in the process described in the embodiment above.The resulting carrierSIS preparation containing a known molar amount ofeach extended SIS peptide is characterized by fluorescencespectrophotometry to determine the amount of fluorescence emission pernanomole of SIS peptide. The amount of carrierSIS to be added isconfirmed either before or after addition to the sample by fluorescenceemission by GFP. Because GFP is extremely resistant to tryptic cleavage,the amount of carrier can also be measured during or after digestion.

8) In another embodiment, multiple carrierSIS products are made in orderto facilitate standardized measurement of proteins having widelydifferent abundances in the sample. Thus, a first carrierSIS productincludes SIS peptide sequences corresponding to tryptic peptides fromproteins having expected concentrations around 1 mg/ml in human plasma(e.g., hemopexin and alpha-1-antichymotrypsin, while a second carrierSISproduct is made containing monitor peptide sequences from low abundance(e.g., 10-1000 pg/ml) proteins such as IL-6 and TNF-alpha. Since themass spectrometer detection systems used to measure the relativeabundances of natural and SIS peptides have limited dynamic range(typically 100 to 10,000), it is desirable to add an amount of each SISpeptide close to the expected amount of the equivalent natural monitorpeptide. Thus, the second carrierSIS described would optimally be addedat a level approximately 1,000,000-fold less than the first carrierSISabove. In cases where the numbers of SIS peptides required inquantitative studies exceed the number that can conveniently be preparedas one carrierSIS protein, it is natural and efficient to group thedesired SIS peptides into classes according to the expectedconcentration of the proteins from which they arise in the sample. If aset of monitor peptides were selected within a decade of concentrationrange (i.e., all members within a factor of 10 in expectedconcentration), then 6 carrierSIS products would be required to span atotal dynamic range of 1,000,000 between the most and least abundanttarget protein. It is also possible to employ carrier polySIS peptidecomplexes where the polySIS peptides contain different SISpeptidesubsequences in different relative proportions to facilitatestandardized measurement of proteins at different abundances (e.g. thesubsequences may be preset at a ratio selected from: 1:20, 1:10, 1:8,1:6, 1:4, or 1:2).

9) In another embodiment, magnetic particles coated withmaleimide-activated protein are used as carrier. In this case an excessof particles (in terms of moles of reactive maleimide) is reacted with aknown molar amount of extended SIS peptide having an n-terminal Cys inthe extension. A known fraction of the resulting preparation in liquidsuspension is added to a protein-containing sample and digested withtrypsin. After digestion, a magnet is used to remove the carrierparticles. The SIS-spiked sample, without the carrier, is analyzed bymass spectrometry.

10) In another embodiment, unequal stoichiometries between two or moreindividual SIS peptides are achieved by coupling a mixture of extendedSIS peptides, wherein each is present at a known molar concentration,with an excess of activated carrier. Thus, a carrierSIS product with 1copy of a SIS sequence denoted A, 2 copies of a SIS sequence denoted B,4 copies of a SIS sequence denoted C and 10 copies of a SIS sequencedenoted D can provide peptide standards at concentrations that match theamounts of monitor peptides derived from proteins expected to be presentat relative concentrations of 1:2:4:10 in the original sample.Alternatively, a carrier bearing a polySIS (carrierPolySIS) sequence inwhich the subsequences A, B, C and D were present in a ratio of 1:2:4:10respectively could achieve essentially the same result and would requirefewer binding sites on the carrier to incorporate the same molar amountof total SIS peptides that would be released into the sample.

11) In another embodiment, two or more monitor peptide sequences areselected from the digest products of a single target analyte protein,and extended SIS sequences for each of these are incorporated into thecarrierSIS product. Thus, SIS sequences A, B and C from a given targetprotein may be incorporated into the carrierSIS at equal molar amounts,or alternatively different molar amounts (e.g., at the upper and lowerconcentrations of a normal range for an analyte protein). Measurement ofmultiple monitor peptides from a single protein target (in which thepeptides are present at defined, usually 1:1:1, stoichiometry) andcomparison to multiple SIS peptides added at accurately defined 1:1:1stoichiometry in a carrierSIS mixture provides improved measurementprecision and allows accurate detection of situations in which only partof a target protein is present. Alternatively, addition of a polySISpeptide or carrierPolySIS conjugate in which the subsequences A, B, andC are present in a ratio of 1:1:1 respectively could achieve the sameincrease in precision, and would require calibration of only oneinternal reference standard.

12) In another embodiment, an easily assayed substituent is incorporatedinto the carrierSIS and used for later quantitation of the carrierSISconjugate. An example is the incorporation of biotin groups. Thepresence of the biotin group at 1 mole per mole of carrierSIS allowsabsolute quantitation of the carrierSIS through use of a standard assayfor the biotin tag (e.g., a competition assay using immobilizedstreptavidin as capture agent and a biotinylated acid phosphatase as thecompeting ligand able to generate a colorimetric signal). In addition,the biotin tag can be used for purification of the bulk carrierSISconjugate (where this is of molecular dimensions) by binding to astreptavidin column. The carrierSIS can be released from such a columnby selective elution or by cleavage at a peptide sequence linking theSIS sequences to the biotinylated site using a specific protease (e.g.,Factor Xa) with a specificity different from the protease used toliberate SIS (e.g., trypsin).

13) In another embodiment, entire domains of target proteins that arelabeled with stable isotopes (e.g., during in vitro or in vivoexpression or chemical synthesis) are coupled to the carrier instead ofshort individual extended SIS peptide sequences. In this approach, eachdomain contains at least one SIS peptide (e.g., tryptic SIS peptide(s)).In those embodiments where one or more domains contain several peptidessuitable for measurement by mass spectrometry (e.g., tryptic SISpeptides), those domains offer multiple opportunities to quantitate thetarget. More importantly, by including entire domains likely to fold ina manner more similar to the fold of part of the intact whole targetprotein, the carrierSIS better replicates the environment within whichthe proteolysis will occur for the native target protein—i.e., thecleavage of the peptides in the carrierSIS may better parallel theefficiency in the target.

14) In another embodiment, one or more SIS peptides are applied in knownamounts to a surface of a single-use component provided in an assay kit,in a form that preserves peptide integrity during kit storage and allowsrelease of the SIS when the surface contacts the sample. For example,three SIS peptides are deposited at the bottom of a well of apolypropylene 96-well plate in a small volume of an aqueous matrixcontaining dissolved trehalose and then dried in place, each in anamount equal to that required to establish a respective pre-determinedtest evaluation threshold in a specified volume (e.g., 100 microliters)of a protein-containing sample such as human plasma or a digest ofplasma. Once the pre-specified volume of applied sample dissolves thedried SIS peptides, they are available to act as internal standards. Avariety of peptide stabilizers are known in the art that are capable ofincorporating or adsorbing SIS peptides in such a way that they readilydissolve in an applied liquid sample. Such surface applied SIS peptidescan be placed in or on tubes, microtiter wells, pipette tips, pins, andother single-use components of an assay kit to provide accurate amountsof internal standard. Use of this approach has the advantage that theamount of SIS delivered per assay is controlled at the point ofmanufacture of the component and does not depend on the accuracy of SISdispensing at the point of use. CarrierSIS conjugates, polySISpolypeptides, or carrierPolySIS conjugates can be similarly placed onsurfaces in place of SIS peptides. Components that are exposed to thedigestion process (e.g., to a proteolytic enzyme) are appropriate sitesfor covalent binding of carrierSIS to which SIS are linked by aproteolytically cleavable linker

Examples

In a first example, a tryptic peptide analyte (having sequenceFSPDDSAGASALLR (SEQ ID No. 4) and protonated monoisotopic mass 1406.691)is selected to allow measurement of human thyroglobulin in plasma.Synthetic peptides having this sequence are generated by conventionalsolid phase peptide synthesis using i) precursors with natural isotopiccomposition (i.e. yielding an unlabeled peptide equivalent to theanalyte) and ii) the same precursors except for use of labeled arginine(whose precursor is substituted with ¹³C and ¹⁵N at all positions),yielding a SIS peptide with a stable isotope labeled c-terminal arginineresidue and hence a mass that is 10 atomic mass units (amu) greater thanthe unlabeled analyte.

A series of dilutions of synthetic analyte peptide are prepared, eachcontaining the same concentration of the SIS version of this analyte, sothat the ratio of analyte to SIS amounts varies across the dilutionseries. In this example, the amount of the SIS version illustrates theclinical decision point of an assay for the peptide. Thus, in thesimplest embodiment described here, the result of the assay is given bycomparing the amount of the analyte to the amount of the SIS andproviding a binary assay result depending upon whether the analyte ispresent in greater or lesser amount than the added SIS here used as aninternal standard at a clinical decision threshold. Two ul (microliters)of peptide samples were applied to MALDI matrix spots on a pre-spottedMALDI target (Eppendorf Pre-spotted Anchor Chip targets sold by BrukerDaltonics) in 5% acetic acid for 3 minutes, followed by 5 ul of ammoniumphosphate in 0.1% trifluoroacetic acid, after which the liquid waswithdrawn and the target dried in air. MALDI spectra were obtained witha Bruker Autoflex MALDI mass spectrometer in reflection mode, andprocessed using Bruker flexAnalysis software to yield centroid peakheights and areas.

FIG. 5 shows the MALDI mass spectra of three samples in the dilutioncurve: A in which the unlabeled analyte peptide (at mass 1406.7) ispresent in an amount greater than the SIS at mass 1416.7, the differencein peak heights being amount 31; B in which the amounts are very nearlyequal; and C in which the analyte peak is less than the SIS peak byamount 32. The ratios of the heights (intensities) of the major analyteto SIS peaks in A, B and C are 1.183, 0.991, and 0.856, withcoefficients of variation of 2.1%, 1.1% and 1.8% across a series ofreplicate MALDI spots (4, 3, and 4 spots respectively). Panel A thusrepresents a case in which the analyte is about 19% greater in intensitythan the SIS, which given an average CV (coefficient ofvariation=standard deviation divided by the mean) in this range of 1.7%,is almost 12 standard deviations above the clinical decision level.Panel C represents a case in which the analyte is about 14% below theintensity of the SIS, which is more than 8 standard deviations below theclinical decision level. If centroid peak area is used instead of peakintensity, the respective ratios are 1.187, 1.003, 0.837. Clearly thedetection scheme illustrated here can unequivocally determine thatanalyte is more or less than the clinical decision level by ˜15% withextreme statistical precision (i.e., more than 8 standard deviations).

Panel B of FIG. 5 represents a case in which the analyte level is veryclose to the clinical decision level (assumed here to be the known levelat which the SIS peptide is added to the sample). A set of 4 MALDItarget spots prepared from each of 3 replicate dilution series (12 totalreplicates) of the composition shown in panel B were analyzed by MALDIand the results for the analyte and SIS peaks obtained. The averageratio over 12 replicates between analyte and SIS peak heights(intensities) was 0.9998 with a CV of 1.5%. The average ratio over 12replicates between analyte and SIS peak areas (calculated by a centroidalgorithm) was 0.9948 with a CV of 1.1%. Any ratio that is more than4.5% above or below a measured ratio of 1.0 would therefore represent asignificance of 3 or more standard deviations from the clinical decisionlevel. This level of result precision in relation to a clinical decisionlevel is better than most current clinical protein immunoassays, both interms of absolute precision of measurement and because of the ability touse internal, rather than external, standardization (contributing tobetter test accuracy).

In a second example, a series of peptide analytes at approximately equalmolar abundances is used to prepare a series of dilutions in relation tofixed concentrations of the SIS versions of the same peptides,illustrating in one embodiment the method applied to a multiplexed testsituation. Analyte peptides 1-5 are measured (as peak intensity) inrelation to SIS peptides 1-5 added as internal standards at theirrespective multiplex test thresholds. Analytes 1 and 4 exceed theirrespective thresholds, while analytes 2, 3 and 5 are lower than theirrespective thresholds. The result of the test is determined bycomparison with a pre-established matrix of outcomes: the result patternHigh-Low-Low-High-Low is looked up in this table and found to generate anegative test result.

In a third example, a “carrierSIS protein” is prepared by linkingextended SIS peptides to an activated protein carrier (as shown in FIGS.6A and B). Carrier protein 1, in this case keyhole limpet hemocyanine(KLH), is activated by modification with multiple groups covalentlyattached to the KLH, each of which comprises a linker segment 2 and amaleimide reactive group 2 (“M”). This modification is accomplished byreaction of purified KLH with4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid3-sulfo-N-hydroxysuccinimide ester (a heterobifunctional crosslinkingreagent that reacts with protein primary amino groups to providesulfhydryl-reactive maleimide groups on the protein surface).

SIS peptides are chemically synthesized with an n-terminal sequenceextension containing a cysteine residue (whose sulfhydryl reacts withmaleimide to yield a stable chemical bond), several spacer amino acids(here -GSG-, SEQ ID No. 1) and a final lysine (K) residue adjacent tothe SIS n-terminus, creating a tryptic cleavage site. Peptide synthesisis typically carried out on a solid phase resin (Merrifield, MethodsEnzymol 289:3-13, 1997), and can include steps to ligate togethermultiple synthetic peptides to produce larger peptides or proteins(Dawson, Muir, Clark-Lewis and Kent, Science 266:776-9, 1994, Dawson andKent, Annu Rev Biochem 69:923-60, 2000). One embodiment makes use ofstable isotope labeled K and R, since each tryptic SIS peptide containsonly one such residue (either K or R) per peptide at the c-terminus,thus simplifying the calculation of mass shifts in the y-ion fragmentseries often used for selected reaction monitoring (SRM) quantitation intandem mass spectrometry. Incorporation of labeled K or R is achievedthrough use of the corresponding labeled K or R synthons commerciallyavailable for solid phase peptide synthesis. Alternatively any aminoacid containing stable isotope labels can be used.

Thus, the SIS peptide “EIGELYLPK*” (SEQ ID No. 5, corresponding to atryptic peptide of human alpha-1-antichymotrypsin and incorporating ac-terminal stable isotope-labeled lysine shown as K* and described asU-¹³C₆, 98%; U-¹⁵N₂, 98%, thus, having a mass 8 amu greater than theunlabeled natural analyte peptide of the same sequence) is synthesizedas the extended SIS peptide sequence “CGSGKEIGELYLPK*” (SEQ ID No. 6).This extended peptide reacts with activated KLH through reaction of thepeptide's n-terminal cysteine sulfhydryl and a maleimide group onactivated KLH. Action of trypsin on this extended SIS sequence bound toKLH cleaves between K and E residues, releasing the SIS sequence“EIGELYLPK*” (SEQ ID No. 5) without any extension and which is capableof functioning as a labeled internal standard for mass spectrometricmeasurement of EIGELYLPK (SEQ ID No. 5) (and thus indirectly ofalpha-1-antichymotrypsin) in a sample digest. Similarly a second SISpeptide NFPSPVDAAFR* (SEQ ID No.7, a tryptic peptide of human hemopexin)is synthesized as the extended SIS “CGSGKNFPSPVDAAFR*” (SEQ ID No. 8)and reacted with activated KLH, combining with other maleimide groups onthe activated KLH molecule. Action of trypsin on the bound extended SISreleases the SIS sequence NFPSPVDAAFR*, (SEQ ID No. 7) capable offunctioning as a labeled internal standard for mass spectrometricmeasurement of sample-derived NFPSPVDAAFR (SEQ ID No. 7) and thus of theparent protein hemopexin.

To form the SIS peptide:carrier conjugate, a known amount of each SISpeptide (as established by quantitative amino acid analysis) is added toa 1.2 fold molar excess of activated KLH carrier (as determined bytitration of available maleimide groups) and allowed to react for 6hours (hr) at room temperature.

A known amount of carrierSIS (i.e., a known volume of standardizedsolution) is then added to a measured volume of a sample in which thetarget proteins are to be quantitated (in this case a sample of humanblood plasma). This combined sample, including spiked carrierSISstandard, is then proteolytically digested by exposure to trypsin usingany of a variety of well-known protocols. In one such protocol, plasmais denatured by addition of 9 volumes of 6 M guanidinium HCl/50 mMTris-HCl/10 mM dithiothreitol and incubation for 2 hr at 60° C.;addition of 1 volume of 200 mM iodoacetamide followed by incubation for30 min at 25 C; addition of 1 volume of 200 mM dithiothreitol followedby incubation for 30 min at 25 C; dilution to <1 M guanidinium HCl byaddition of 50 mM NaHCO₃, addition of sequencing grade modified trypsin(e.g., from Promega, Madison, Wis.) at a 1:50 ratio (trypsin:plasmaprotein) and incubation overnight at 37° C. Digestion is allowed toproceed until substantially complete, liberating the monitor peptidesfrom both target proteins and carrierSIS protein essentially tocompletion. Alternatively a mixture of SIS resulting from priordigestion of carrierSIS protein can be added to the sample before orafter sample digestion. This sample digest now contains versions ofanalyte (monitor) peptides containing natural isotopes (from peptidesderived from the original sample) and stable isotopes (in the SISpeptides derived from the carrierSIS protein). Each sample-derivedanalyte monitor peptide can then be quantitated by measuring itsconcentration relative to the stable isotope version (which has a knownconcentration calculable from the amount spiked into the sample orsample digest) in a mass spectrometer, the results of which (ananalyte:SIS ratio) then allows calculation of the concentration of theassociated target protein in the initial sample (as described inpublished U.S. Pat. No. 7,632,686, High Sensitivity Quantitation OfPeptides By Mass Spectrometry, Anderson, Norman L.). In someembodiments, the relative concentrations of natural and stable isotopelabeled monitor peptides can be measured by mass spectrometry as therelative ion currents recorded as peak intensity or area for the twopeptides using electrospray-MS, or as peak intensity or area byMALDI-MS. The two versions of the peptide perform essentiallyidentically in any chromatographic or affinity based separation orenrichment process (providing the elements N, C or O are used as stableisotope labels), and thus co-elute, facilitating direct comparison ofion currents. In this embodiment, one carrierSIS protein can replace anentire collection of separate SIS peptides described in earlierdisclosures, and eliminates the requirement to separately store andhandle the various SIS peptide reagents. Quantitative MS measurementscan be made using a variety of ionization sources (e.g., electrosprayionization [ESI] and matrix-assisted laser desorption ionization[MALDI]) and mass analyzers (e.g., time-of-flight [TOF], triplequadrupole [TQMS], Fourier transform ion cyclotron resonance [FTICR],and ion trap).

The foregoing disclosure outlines a number of embodiments in terms ofthe SISCAPA method, and therefore represents one set of embodiments thatmay be employed in the application of the present technology. It will beappreciated that the methods and compositions disclosed herein are notlimited to the SISCAPA method, but may be applied to other methods thatemploy internal peptide standards and the like.

1. A method of standardizing a quantitative mass spectrometric assay fora peptide analyte in a sample, comprising: adding a known amount of aSIS version of said analyte to a known amount of said sample, to form astandardized sample, measuring by mass spectrometry the relative amountsof said peptide analyte and said SIS version of said analyte in saidstandardized sample, and comparing the amount of said peptide analyte tothe amount of said SIS version of said analyte, wherein said knownamount of said SIS version of said analyte corresponds to the analyteamount at a pre-determined test evaluation threshold.
 2. The method ofclaim 1, wherein said test evaluation threshold is a clinical decisionthreshold derived from a clinical study establishing test sensitivityand specificity at said threshold. 3-7. (canceled)
 8. A method ofstandardizing a mass spectrometric assay for a protein or peptideanalyte in a sample provided by a donor comprising: adding a knownamount of a SIS version of said analyte as an internal standard to aknown amount of said sample prior to mass spectrometric analysis;wherein said known amount of said internal standard corresponds to ananalyte amount computed from one or more previous measurements of saidanalyte in one or more samples from said donor.
 9. A method ofstandardizing a mass spectrometric assay for a protein or peptideanalyte in a sample comprising: adding known amounts of a first SISversion of said analyte and a second SIS version of said analyte asinternal standards to a known amount of said sample prior to massspectrometric analysis; wherein said known amount of said first SISversion corresponds to the analyte amount at the lower end of theestablished clinical reference range for the analyte and said knownamount of said second SIS version corresponds to the analyte amount atthe upper end of the established clinical reference range for theanalyte; and wherein said first and said second SIS versions differ fromthe analyte and from one another in mass and, said first and said secondinternal standards and said analyte have substantially identicalchemical structures.
 10. A method according to claim 1, furthercomprising: adding a known amount of a second SIS version of a secondprotein or peptide analyte in said sample as an internal standard to aknown amount of said sample prior to mass spectrometric analysis;wherein said known amount of said second SIS version corresponds to aclinical reference threshold for said second analyte. 11-13. (canceled)14. A method according to claim 1, further comprising assigning a testresult that differs depending on whether the measured amount of analyteis greater than or less than the measured amount of said SIS version ofsaid analyte; wherein said SIS version of said protein or peptideanalyte differs from the analyte in mass and said SIS version and saidanalyte have substantially identical chemical structures.
 15. The methodof claim 14, wherein said SIS version of said analyte is added to saidsample before measurement in an amount substantially equal to apre-determined test evaluation threshold. 16-18. (canceled)
 19. A methodaccording to claim 10, further comprising assigning a test result thatdiffers for each analyte depending on whether the measured amount of theanalyte is greater than or less than the measured amount of the SISversion of that analyte; wherein each of said SIS versions of said twoor more peptide or protein analytes has substantially identical chemicalstructure to the analyte for which it serves as a standard but differsfrom that analyte in mass.
 20. (canceled)
 21. A method according toclaim 9, further comprising adding known amounts of first and second SISversions of a second peptide or protein analyte to said sample; ii)measuring the amount of each of said peptide or protein analytes andeach of said first and second SIS versions of each of said peptide orprotein analytes by mass spectrometry; iii) for each of said peptide orprotein analytes, comparing the measured amount of each analyte with themeasured amount of each of said first and second SIS versions of thatanalyte in said sample; and iv) assigning a test result that differs foreach of said peptide or protein analytes depending on whether the amountof the each analyte is greater than, less than, or in a range betweenthe amount of the first and second SIS versions of that analyte added tothe sample wherein, the first version of each of said analytescorresponds to a lower analyte limit for each of said analytes; wherein,the second versions of each of said analytes corresponds to an upperanalyte limit for each of said analytes; and wherein said first andsecond versions of each of said peptide or protein analytes havesubstantially identical chemical structure to the analyte for which theyserves as standards but differ in mass from that analyte and each other.22. (canceled)
 23. A method for the evaluation of the result of amultiplexed test for a panel of different peptide or protein analytes ina sample, said method comprising the steps of i) comparing a measuredamount of each analyte with a measured amount of a corresponding SISversion of said analyte in said sample to obtain a greater than orlesser than binary decision for each analyte, and ii) combining saidbinary decisions for each of the analytes in said panel according to apre-specified table, wherein each possible combination of binary analyteresults is linked to a pre-specified result for the multiplexed test,wherein each of said corresponding SIS versions of said analyte hassubstantially identical chemical structure to the analyte for which itserves as a standard, but differs in mass from that analyte. 24-30.(canceled)
 31. A carrierSIS complex comprising: a carrier having carrierlinkage sites and at least one extended SIS peptide having a peptidelinkage site; wherein said carrier and said peptide are bound through alinkage between said peptide linkage site and said carrier linkagesites, wherein said SIS peptide can be liberated from said carrier byaction of a proteolytic process yielding a free SIS peptide, and whereinsaid SIS peptide contains at least one site at which a non-predominantstable isotope label is substituted for the predominant naturallyoccurring isotope of the atom appearing at that site in more than 95% ofthe SIS peptides bound to said carrier.
 32. (canceled)
 36. ThecarrierSIS complex of of claim 31, wherein multiple different SISpeptides and/or extended SIS peptides are bound to said carrier, andwherein the multiple different SIS peptides and or extended SIS peptidesare optionally bound to said carrier at different relativeconcentrations.
 37. The carrierSIS complex of claim 31, wherein thecarrier further comprises a moiety that can be assayed byspectrophotometry, fluorescence, mass spectrometry, an assay for thepresence of biotin, or a moiety whose presence can measured by enzymeassay, permitting an amount of carrier to be measured.
 38. ThecarrierSIS complex of claim 31, wherein the carrier has one or morecarrier quantitation peptides attached to it that can be released duringproteolytic digestion, and which carrier quantitation peptides whenreleased from said carrier permit quantitation of the carrier itselfversus the SIS peptides bound to the carrier. 39-47. (canceled)